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Meng Chen, Terry Coursey, Johanna Tukler Henriksson, Cintia De Paiva, De-Quan Li, Stephen Pflugfelder; Increased proliferation of cultured conjunctival epithelium in IFN-γ deficient strains. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2076.
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© ARVO (1962-2015); The Authors (2016-present)
Previous experiments have demonstrated that IFN-γ promotes conjunctival epithelial apoptosis and goblet cell loss. The objective of this study was to compare epithelial growth and differentiation in conjunctival epithelial cultures initiated from IFN-γ deficient mice and wild type (WT) C57BL/6 mice.
Primary conjunctival epithelial cultures were established in parallel from fresh tissue explants of forniceal conjunctiva dissected from IFN-γ gene knockout (IFN-γKO) and WT C57BL/6 age- and gender-matched mice. Cultures were incubated with Keratinocyte SFM (KSFM) medium supplemented with 80 ng/mL murine EGF and 3% fetal bovine serum. Cell proliferation was measured by WST-1 assay. Explant outgrowth areas were quantified using Celltrace Calcein Green AM staining and Nikon Elements software analysis. Keratin 7 (K7) and MUC5AC were immunodetected by immunofluorescent staining. K7 and MUC5AC mRNA expression were measured by quantitative real time PCR.
Seventy-seven percent of IFN-γKO cultures reached 70-80% confluence after 2 weeks, compared to 61% of WT C57BL/6 cultures. Positive K7 and MUC5AC immunofluorescent staining confirmed the growth of conjunctival epithelium and goblet cells in culture. WST-1 cell viability assay showed IFN-γKO cultures had 2.24 fold (p<0.05, n=9) and 2.01 fold (p<0.05, n=6) greater number of viable cells at 7 and 14 days, respectively, compared to WT C57BL/6 cultures. Calcein Green AM staining also demonstrated statistically significant increased epithelial outgrowth from IFN-γKO explants compared to C57BL/6 . At 4 days, mean growth area of WT C57BL/6 and IFN-γ cultures were 1.52 x 107 µm and 2.80 x 106 µm (p<0.05, n=5), respectively, and at 7 days, they were 2.33 x 107 µm and 6.14 x 106 µm (p<0.05, n=4), respectively. A significant 2.58 fold elevation in MUC5AC mRNA transcripts was observed in 2 week old IFN-γKO cultures compared to WT C57BL/6 cultures, but K7 mRNA transcripts were not significantly different between the two strains.
Our findings show that cultured conjunctival epithelial and goblet cells from IFN-γ deficient mice have increased growth and differentiation compared to wild-type mice. This suggests IFN-γ suppresses conjunctival epithelial growth and goblet cell differentiation.
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